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BACKGROUND: Recently, suspected cyhalofop‐butyl‐resistant populations of allohexaploid weed Echinochloa crus‐galli var. crus‐galli were discovered in rice fields in Aichi Prefecture, Japan. Analyzing the target‐site ACCase genes of cyhalofop‐butyl helps understand the resistance mechanism. However, in E. crus‐galli, the presence of multiple ACCase genes and the lack of detailed gene investigations have complicated the analysis of target‐site genes. Therefore, in this study, we characterized the herbicide response of E. crus‐galli lines and thoroughly characterized the ACCase genes, including the evaluation of gene mutations in the ACCase genes of each line. RESULT: Four suspected resistant lines collected from Aichi Prefecture showed varying degrees of resistance to cyhalofop‐butyl and other FOP‐class ACCase inhibitors but were sensitive to herbicides with other modes of action. Through genomic analysis, six ACCase loci were identified in the E. crus‐galli genome. We renamed each gene based on its syntenic relationship with other ACCase genes in the Poaceae species. RNA‐sequencing analysis revealed that all ACCase genes, except the pseudogenized copy ACCase2A, were transcribed at a similar level in the shoots of E. crus‐galli. Mutations known to confer resistance to FOP‐class herbicides, that is W1999C, W2027C/S and I2041N, were found in all resistant lines in either ACCase1A, ACCase1B or ACCase2C. CONCLUSION: In this study, we found that the E. crus‐galli lines were resistant exclusively to ACCase‐inhibiting herbicides, with a target‐site resistance mutation in the ACCase gene. Characterization of ACCase loci in E. crus‐galli provides a basis for further research on ACCase herbicide resistance in Echinochloa spp. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Hybridization is a major source of phenotypic variation and a driving force for evolution. Although novel hybrid traits can often disrupt adaptive relationships between the parental phenotypes and their environments, how new hybrid traits disrupt local adaptation remains unclear. Here, we report how a new phenotype of hybrids between two Imperata cylindrica ecotypes contributes to rapid reproductive isolation from their parents and affects hybrid fitness.We analysed 350 accessions of I. cylindrica collected from the 1980s to the 2010s throughout Japan to explore the genetic population structure of the hybrids. We surveyed the flowering periods, seed set, and germination of two ecotypes and their hybrids in both natural habitats and common gardens.Genetic analyses of population structure revealed that the hybrid populations consisted of only F1 individuals, without advanced generation hybrids. The flowering phenology of the F1 plants was delayed until autumn, 5–6 months later than the parental ecotypes. The drastic shift in flowering phenology prevents F1s from backcrossing. In addition, it changes their seed dispersal time to winter. Germination is inhibited by low temperatures, and the seeds likely decay before the next spring, resulting in the absence of an F2 generation. We identified the environmental mismatch of the F1 population as a specific mechanism for the maintenance of an only F1 population.Synthesis. We have demonstrated that this flowering phenology mismatch promotes reproductive isolation between the parents and F1s and affects various temporal components of the hybrids, resulting in a unique hybrid population consisting only of F1s. This system sheds light on the importance of hybrid traits in driving rapid reproductive isolation. [ABSTRACT FROM AUTHOR]

Herbicides are often selective and kill only weeds without affecting crops. In rice, CYP81A6 is responsible for tolerance toward acetolactate synthase herbicides such as bensulfuron‐methyl (BSM). CYP81A6 is thought to metabolize BSM into a nontoxic metabolite, that is, O‐demethylated BSM. However, direct evidences for this reaction have not been reported, likely due to difficulties in the heterologous expression of plant P450s in bacterial expression systems. In this study, we established CYP81A6 expression in Escherichia coli via modification of its N‐terminal basic region coding sequence, optimization of cultivation temperature, and selection of E. coli strains. Heterologous production of CYP81A6 was evaluated by the conversion of BSM in autoinduction medium after the culture, compared to that with an empty vector control. The optimized heterologous production condition of rice CYP81A6 in E. coli using 48‐well plates involved replacement of the N‐terminal basic region with that of CYP2C3, the use of E. coli BL21(DE3) as an expression host, and culture at 34°C. Further, co‐expression of gene encoding glucose dehydrogenase enhanced the conversion of 0.5 mM BSM from 58.7 to 73.1% in the optimized condition. In the optimization process, we detected two metabolites, M1 and M2. Mass spectrometry and 1H nuclear magnetic resonance analyses indicated that M1 was O‐demethylated BSM. Although we could not isolate M2 because of its low abundance in the medium, mass spectrometry analysis suggested that it was the monooxygenated (+O) form of BSM. Overall, these results confirm that CYP81A6 catalyzes the formation of O‐demethylated BSM from BSM. [ABSTRACT FROM AUTHOR]

Summary: Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase‐type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)‐mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR‐mediated GT is extremely rare in higher plants, positive–negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac‐transposon‐mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I‐SceI break and subsequent single‐strand annealing (SSA)‐mediated DNA repair system. [ABSTRACT FROM AUTHOR]

Cytochrome P450s (P450s) have been at the center of herbicide metabolism research as a result of their ability to endow selectivity in crops and resistance in weeds. In the last 20 years, ≈30 P450s from diverse plant species have been revealed to possess herbicide‐metabolizing function, some of which were demonstrated to play a key role in plant herbicide sensitivity. Recent research even demonstrated that some P450s from crops and weeds metabolize numerous herbicides from various chemical backbones, which highlights the importance of P450s in the current agricultural systems. However, due to the enormous number of plant P450s and the complexity of their function, expression and regulation, it remains a challenge to fully explore the potential of P450‐mediated herbicide metabolism in crop improvement and herbicide resistance mitigation. Differences in the substrate specificity of each herbicide‐metabolizing P450 are now evident. Comparisons of the substrate specificity and protein structures of P450s will be beneficial for the discovery of selective herbicides and may lead to the development of crops with higher herbicide tolerance by transgenics or genome‐editing technologies. Furthermore, the knowledge will help design sound management strategies for weed resistance including the prediction of cross‐resistance patterns. Overcoming the ambiguity of P450 function in plant xenobiotic pathways will unlock the full potential of this enzyme family in advancing global agriculture and food security. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND: Multiple herbicide resistant Echinochloa phyllopogon exhibits resistance to the auxin herbicide quinclorac. Previous research observed enhanced activity of the cyanide‐detoxifying enzyme β‐cyanoalanine synthase (β‐CAS) and reduced ethylene production in the resistant line, suggesting β‐CAS‐mediated cyanide detoxification and insensitivity to quinclorac stimulation as the resistance mechanisms. To investigate the molecular mechanisms of quinclorac resistance, we characterized the β‐CAS genes alongside plant transformation studies. The association of β‐CAS activity and ethylene production to quinclorac resistance was assayed in the F6 progeny of susceptible and resistant lines of E. phyllopogon. RESULTS: A single nucleotide polymorphism in a β‐CAS1 intron deleted aberrantly spliced mRNAs and enhanced β‐CAS activity in the resistant line. The enhanced activity, however, was not associated with quinclorac resistance in F6 lines. The results were supported by lack of quinclorac resistance in Arabidopsis thaliana expressing E. phyllopogon β‐CAS1 and no difference in quinclorac sensitivity between β‐CAS knockout and wild‐type rice. Reduced ethylene production co‐segregated with quinclorac resistance in F6 lines which were previously characterized to be resistant to other herbicides by an enhanced metabolism. CONCLUSION: β‐CAS does not participate in quinclorac sensitivity in E. phyllopogon. Our results suggest that a mechanism(s) leading to reduced ethylene production is behind the resistance. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]